Anti-tumor protein peptide capable of inhibiting FOXM1

A protein peptide and anti-tumor technology, applied in the fields of genetic engineering and oncology, can solve the problem of inability to obtain induced pluripotent stem cells

Active Publication Date: 2018-03-23
XINSHENG KANGYUAN BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, FOXM1 is involved in the maintenance of cell stemness (Nucleic Acids Res 2010. 38: 8027-8038). Inhibition of FOXM1 leads to the inability t...

Method used

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  • Anti-tumor protein peptide capable of inhibiting FOXM1
  • Anti-tumor protein peptide capable of inhibiting FOXM1
  • Anti-tumor protein peptide capable of inhibiting FOXM1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, Construction of prokaryotic expression vector pHis-M1-138-R9.

[0036] 1. Amplification of the His-M1-138-R9 gene fragment. Design primers to amplify the gene fragment by PCR. The primer sequence is: Primer 1 (upstream primer): GCG CCC ATG GTG CAT CAC CAT CAC CAT CAC ATG AAA ACT AGC CCC CGTCG, primer 2 (downstream primer): GCG GGA TCC CTA CCT TCT CCT TCT CCT TCT CCT TCT CCT CAGGGT CAC TTC TGT C. Using pcDNA3.1-FOXM1 as a template, under the guidance of primers 1 and 2, the reaction system for PCR amplification of M1-138 was as follows: clone plasmid pcDNA3.1-FOXM1 (80ng / μL) 1 μL, 10X PCR Buffer (ThermoScientific) 5 μL, dNTPs (2mM each) 5μL, MgSO 4 Solution (25mM) 4μL, KOD-Plus-Neo (1.0U / μL) 1μL, primer 1 (100μM) 1μL, primer 2 (100μM) 1μL, DMSO 2μL, add deionized water to supplement the reaction system to 50μL. PCR reaction conditions: first 94°C for 5min; then 95°C for 30sec, 57°C for 30sec, 68°C for 50sec, a total of 30 cycles; then 68°C for 10min, 4°C fo...

Embodiment 2

[0039] Example 2, large-scale purified M1-138 recombinant protein.

[0040] 1. Induced expression of M1-138 recombinant protein. The prokaryotic expression vector pHis-M1-138-R9 was transformed into Escherichia coli Rostta DE3 competent cells, cultured at 37°C overnight (12-16hr), randomly selected single clones, and inoculated into 5mL LB medium (containing 25μg / mL ampicillin and 25μg / mL chloramphenicol), shake culture at 37℃ for 6-8hr. Add the bacteria solution to 100 mL of LB culture solution (containing 25 μg / mL ampicillin and 25 μg / mL chloramphenicol) and shake at 37°C overnight (12-16hr), take the bacteria solution to detect the OD 600 value, adjust OD 600 When the value reached 0.8-1, IPTG inducer (final concentration 8 μM) was added, and shaking culture was induced at 20°C for 20 hr. Centrifuge at 4000rpm for 20min to collect the bacteria, and use 15mL Binding Buffer (20mM Na 3 PO 4 , 500mM NaCl, 20mMimidazole, pH 7.4) to resuspend the bacteria, and sonicate for 4...

Embodiment 3

[0042] Example 3, M1-138 recombinant protein inhibits lung cancer cell line A549.

[0043] In order to verify the inhibitory effect of M1-138 on the proliferation of lung cancer cells, lung cancer A549 cells were selected, and the commercial Cell Counting Kit (CCK) kit was used to investigate the effect of M1-138 on the proliferation of tumor cells. Seed the cell suspension (100 μL, 4000 cells / well) in a 96-well plate. Place the culture plate in the incubator (37°C, 5% CO 2) after pre-incubation for 12 hr, the cells were treated with different concentrations of M1-138 (0.5, 1, 2, 4, 8, 16, 20, 24, 28, 32, 36 μM) and cultured for another 12 hr. Add 10 μL of CCK WST-8 reagent to each well, incubate in the incubator for 1-4hr, measure the absorbance at 450nm with a microplate reader, and calculate the cell viability of the cells to be tested, cell viability*(%)=[A( Added drug)-A (blank)] / [A (no drug added)-A (blank)] ×100, where, A (medicated): absorbance of cells, CCK solution...

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Abstract

The invention discloses a protein peptide fragment sourced from FOXM1 protein and containing 1-138-position amino acid residue sequences at a nitrogen end of the FOXM1 protein, and provides a candidate capable of inhibiting functions of FOXM1 and used for developing protein peptide type anti-tumor drugs. The protein peptide fragment contains protein with one of the following amino acid residue sequences: 1) a protein shown in SEQ ID NO:1 in a sequence table; 2) a protein peptide capable of inhibiting or reducing activity and functions of FOXM1 by replacing, deleting, inserting and/or adding one, two or more amino acid residues to the protein shown in SEQ ID NO:1 in the sequence table. The recombinant protein prepared on the basis of the amino acid sequence shown in SEQ ID NO:1 in the sequence table and having membrane penetrating capacity shows an inhibition action on various tumor cells.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and oncology, and relates to the expression of an anti-tumor protein peptide that inhibits FOXM1 and its use. Background technique [0002] From the gene expression analysis of various human tumor samples, it was found that the expression of transcription factor FOXM1 was increased in tumor cells, and the detection of its expression level has been used in the diagnosis and prognosis of various tumors (US7056674, 7081340, 7308364, 7526387, 7531300, CN201510355817 .5). From the perspective of gene function, FOXM1 was first identified as a protein regulating cell cycle and cell proliferation (Mol Cell Biol 1997. 17: 1626-1641). In the process of cell proliferation, FOXM1 is involved in regulating the transcription of multiple genes related to the cell cycle, thereby controlling the process of cell DNA replication and mitosis (Mol CellBiol 1999. 19: 8570-8580, Proc Natl Acad Sci US A 2002. 99: 168...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N15/63C12N15/62A61K38/17A61P35/00
CPCA61K38/00C07K14/4748C07K2319/10C12N15/63C12N15/70
Inventor 谭拥军张振旺余景卫
Owner XINSHENG KANGYUAN BIOPHARML
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